Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have isolated Escherichia coli transcription complexes, paused in the presence and absence of Nus A, which contain RNA substituted at every UMP residue with a photocrosslinking nucleotide analog. The pause site is immediately downstream from an RNA stem-loop structure, and although pausing occurs in the absence of Nus A, it is substantially enhanced in the presence of Nus A. We have analyzed the secondary structure of this RNA and show that the analog does not interfere with the formation of the normal stem-loop structures. Additionally, the analog substrate does not alter transcriptional pausing, in the presence or absence of Nus A, indicating that Nus A recognition of the transcription complex is not affected by the presence of the crosslinking groups in the RNA. Ribonuclease digestion of the RNA in paused complexes identifies two accessible regions, two nucleotides in the loop and one near the base of the upstream side of the stem-loop. Cleavage at one loop nucleotide is enhanced by Nus A, while the nucleotide near the base of the stem-loop is partially protected. Upon irradiation of the transcription complex, Nus A is not photoaffinity labeled by the RNA, even at a high molar ration to RNA polymerase (250:1). Both the beta and beta' subunits are labeled, however, indicating that the putative stem-loop binding domain on the core polymerase involves both subunits. Because the nucleotide protected from ribonuclease by Nus A is very near two analogs, yet Nus A is not crosslinked to the RNA, it is unlikely that Nus A could be protecting this position through direct contact. Furthermore, analog is substituted at positions in both the loop and at several positions in the stem, and again, no crosslinking to Nus A is observed. We conclude that enhancement of pausing by Nus A probably does not require direct interaction with the bases in the RNA stem-loop.
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PMID:RNA-protein interactions in a Nus A-containing Escherichia coli transcription complex paused at an RNA hairpin. 170 33

To assess whether myoglobin adversely affects renal adenylate pools, rats were infused with purified myoglobin (50 mg/100 g body wt) for two hours and renal ATP, ADP, and AMP levels were measured in the absence of shock, after 25 minutes of hemorrhagic shock (55 to 60 mm Hg) or 30 minutes post-recovery. In the absence of shock, myoglobin lowered ATP by 24% (assessed 65 min post-infusion) without affecting renal blood flow (RBF). This effect was completely blocked by deferoxamine (DFO) treatment and it could not be reproduced by ribonuclease infusion (a non-Fe containing, but filtered, protein). Myoglobin + shock caused a three- to fourfold greater decline in ATP than did shock alone despite comparable RBFs. Shock plus myoglobin, but neither one alone, induced substantial S1/S2 proximal tubular morphologic damage and a severe reduction in creatinine clearance, confirming synergistic injury. Ribonuclease completely reproduced myoglobin's effect on shock-induced adenylate profiles. DFO +/- hydroxyl radical scavenger therapy (Na benzoate) did not block the myoglobin shock effect on adenylate pools. Post-shock adenylate recovery was not compromised by myoglobin pre-treatment. If renal artery occlusion (RAO), rather than shock, was used as the ischemic challenge, myoglobin had no discernible impact on adenine nucleotide content. This study concludes that: 1) myoglobin modestly lowers baseline adenylate pools due to an Fe dependent mechanism; 2) myoglobin drastically accentuates shock-induced adenylate depletion by a non-hemodynamic/non-Fe dependent mechanism; 3) myoglobin nephrotoxicity cannot be attributed solely to tissue iron loading; and 4) the RAO model can completely mask important influences on ischemic cellular energetics.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Myoglobin depletes renal adenine nucleotide pools in the presence and absence of shock. 200 25

Ribonuclease T1 and the mutant enzymes were cocrystallized with several ribonucleotides, including non-hydrolyzable substrate analogs of di- and triribonucleotides, which have a novel guanylate in which the 2'-hydroxyl group of the ribose is replaced by a fluorine atom. One of the mutant enzymes has a tryptophan residue, instead of Tyr45 of the wild-type enzyme, to enhance the binding of ribonucleotides to the enzyme and the other mutant enzyme has histidine and aspartate residues, instead of Asn43 and Asn44, respectively, to reproduce the natural substitutions found in ribonuclease Ms. Polymorphism of the crystals was observed for wild-type and mutant enzymes. However, orthorhombic crystals, which are virtually all isomorphous to each other, were successfully obtained from wild-type and mutant (Y45W) enzymes by the macroscopic seeding technique using mother crystals of the wild-type ribonuclease T1 complexed with 2'GMP or 3'GMP. The diffraction patterns of these crystals extend beyond 2.5 A resolution and the diffraction data were collected from some of the crystals on a diffractometer up to a range of 2.5 to 1.8 A resolution.
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PMID:Crystallographic characterization of wild-type and mutant ribonuclease T1 complexes with several ribonucleotides. 208 29

Ribonuclease activity in HeLa cell nuclei is markedly inhibited by ADP-ribosylation following incubation of intact isolated nuclei with [14C]NAD. Time course experiments demonstrate that [14C] incorporation into proteins is accompanied by a 50% inhibition of ribonuclease activity on single-strand and double-strand polynucleotides. Inhibition does not occur when 3-aminobenzamide, a potent (ADP-ribose) polymerase inhibitor, is present. Two enzymatic activities that degrade double-strand polynucleotides have been purified and partially characterized. A relevant level of radioactivity resulting from [14C]NAD incubation of nuclei was associated to the purified enzyme. The RNase F1 component, which shows maximal activity on polyU-polyA is demonstrated to be the major ADP-ribose acceptor protein.
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PMID:In vitro inhibition of HeLa cell nuclear ribonucleases by ADP-ribosylation. 211 91

The 77-nucleotide OOP antisense RNA of bacteriophage lambda complements lambda cII-O mRNA in a region that includes 55 nucleotides at the 3' end of the cII gene and 22 nucleotides in the intercistronic region between the cII and O genes. OOP RNA, produced from multicopy plasmids, inhibits lambda cII gene expression by approximately 100-fold through an RNase III-dependent mechanism. Using primer extension analysis of cellular RNA isolated from an induced lambda lysogen that contains an OOP DNA plasmid, we have identified a cleavage site in cII-O mRNA within the region of complementarity with OOP RNA, at 13 nucleotides from the 3' end of that region. Ribonuclease protection experiments demonstrate that almost all cII-O mRNA in this overlap region is cleaved when OOP RNA is overproduced in RNase III+ cells but not in RNase III- cells. RNA fragments are detected that extend into the O gene from the cleavage sites, while the sister fragments that extend into the cII gene cannot be detected and must be eliminated by additional hydrolytic events. Differences in levels of uncleaved mRNA between RNase III+ and RNase III- cells are much less at several hundred nucleotides to either side of the target region. An alternate OOP RNA-dependent hydrolytic process occurs in RNase III- cells that results in cleavages in one of two regions, one close to the cleavage site observed in RNase III+ cells, and the second several nucleotides beyond the end of the complementary region between OOP RNA and cII-O mRNA. In this latter case, the fragments that extend into the cII gene are stable, while the sister O gene fragments are destroyed, in direct contrast to the RNase III-dependent process.
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PMID:RNase III-dependent hydrolysis of lambda cII-O gene mRNA mediated by lambda OOP antisense RNA. 214 37

Increasing the flexibility of a protein enhances its susceptibility to defined proteases in vitro. To ascertain whether flexibility also affects protein stability in vivo, radioiodinated proteins with similar structures, but dissimilar flexibilities, were introduced into HeLa cells using red cell-mediated microinjection. Intracellular proteolysis was then measured as the rate of release of 125I-tyrosine into the medium. Ribonuclease A was considerably more resistant to degradation by purified proteases or in reticulocyte lysate than its flexible derivatives ribonuclease S and S-protein. In contrast, all three proteins were equally stable within HeLa cells. Like the results obtained for RNases, the rates of degradation of trypsin inhibitors, trypsin analogs, and their complexes correlated with flexibility in reticulocyte lysate. However, the intracellular half-lives of anhydrotrypsin and various proteinaceous trypsin inhibitors were not affected upon formation of enzyme-inhibitor complexes. Furthermore, trypsinogen was degraded more slowly than the structurally similar anhydrotrypsin in HeLa cells, although trypsinogen has additional segmental flexibility in its activation domain. Electrophoretic analyses revealed that trypsin-inhibitor complexes remained intact following injection into HeLa cells, and that neither free inhibitors nor anhydrotrypsin formed Triton-stable complexes with soluble cytoplasmic proteins. The observation that the components of the trypsin-inhibitor complexes were degraded simultaneously indicates that neither constituent unfolded prior to the onset of proteolysis. These studies provide evidence that RNases, trypsin, and trypsin inhibitors are degraded by an intracellular proteolytic pathway(s) which recognizes surface features of the folded proteins.
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PMID:Degradation of proteins microinjected into HeLa cells. The role of substrate flexibility. 243 Sep 58

Ribonuclease T1 (RNase-T1) from Aspergillus Oryzae cleaves ribonucleic acid specifically at guanosine to yield oligonucleotides with terminal guanosine-3'-phosphate. It forms a complex with vanadate (association constant K approximately 145 +/- 30 M-1; delta (51V) = -514 ppm) with spectral features similar to the less stable complexes obtained with di- and tripeptides (Gly-His, Pros-His-Ala, Gly-His-Lys, Val-Glu) containing amino acids that are constituents at the active site of the enzyme. Guanosine also forms a (sparingly soluble) complex with vanadate. Its role is mimicked by inosine, which yields two soluble complexes with vanadate, characterized by delta values of -511 (K = 94 M-1) and -523 ppm (K = 305 M-1 in TRIS buffer and 685 m-1 in buffer-free solution). Comparison with literature values leads to an assignment of the delta = -523 signal to a complex where monovanadate, possibly in a trigonal bipyramidal geometry suggested for the transition state of the phosphate analogue, is coordinated to the 2'- and 3'-oxygens of the ribose ring. A drastic increase of complex stability is observed in the ternary vanadate (12-16 mM)/inosine(10.5 mM)/RNase-T1(5.4 mM) system. An approximate lower limit for the association constant is 1.5.10(5) M-2. The spectral characteristics of the main component of the binary vanadate/inosine complex are essentially maintained (delta = -525 ppm, half-width = 960 Hz), suggesting vanadate binding to the enzyme through hydrogen bonds.
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PMID:Binding of vanadate (V) to ribonuclease-T1 and inosine, investigated by 51V NMR spectroscopy. 251 77

A detailed study of the NH resonances of Ribonuclease-S-peptide (1-19 N-terminal fragment of Ribonuclease A) has been carried out in H2O, pH 3.0, in the temperature range 1-31 degrees, and ionic strength 0-1 M. Individual assignments of all NH amide signals have been achieved by means of extensive double resonance experiments. The folding of S-peptide at low temperature has been monitored by examination of the several NH resonance parameters: first, the nonlinearity of chemical shift vs. temperature plots; second, the selective broadening observed for signals assigned to residues 3-13; and third, the decrease of 3JHNCH coupling constants belonging to this region of the polypeptide chain. All these results are in agreement with the formation of a folded structure at low temperature, which is similar to the one found for the S-peptide in the RNase S crystal.
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PMID:NH resonances of Ribonuclease S-peptide in aqueous solution. Low temperature n.m.r. study. 257 22

Though the substantial part of serum ribonuclease (EC 3.1.27.5) is of pancreatic origin, there are no consistent data on changes in activity of serum alkaline ribonuclease in acute pancreatitis. The recent findings suggest that the increase in ribonuclease activity refers only to patients with necrotic outcome of acute pancreatitis. The aim of this study was to reevaluate the suggestion that elevated ribonuclease activity is specifically related to pancreatic necrosis. Our studies included 57 patients with verified acute pancreatitis, and 11 patients evolving haemorrhagic or necrotic lesions of the pancreas. It was found, that the enzyme increasing in some percentage of patients with acute pancreatitis is the Poly-C avid "pancreatic" ribonuclease. This enzyme begins to increase in the 2nd or third day after onset of the disease, always after decrease in serum amylase activity down to levels close to normal range. Ribonuclease activity increased up to days 5 or 6 of the disease, and then decreased along with diminution of disease symptoms upon treatment. Correlation studies showed that increased ribonuclease activity in acute pancreatitis is related to a higher than the 2nd degree of severity of the clinical course of the disease, to pancreatic necrosis, death, diminished glomerular filtration rate, and age. Thus, pancreatic necrosis is not the exclusive factor directing the increased ribonuclease activity in acute pancreatitis, but the increased ribonuclease activity seems to be a late marker of acute pancreatitis of a severe clinical course.
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PMID:Determination of ribonuclease activity in serum of patients with pancreatic necrosis. An attempt of extending the enzyme diagnosis of acute pancreatitis. 261 34

We have used the technique of ribonuclease protection to define genomic variation among circulating isolates of subgroup A respiratory syncytial (RS) virus. RNAs extracted from HEp-2 cells infected with strains to be analyzed were hybridized with a 32P-labeled RNA probe corresponding to the RS virus G glycoprotein (A2 strain). Areas of nonhomology were detected by cleavage with ribonuclease A. Using this technique, multiple distinct RNA cleavage patterns could be distinguished among viral isolates recovered from infants residing in the same metropolitan area and infected during the same epidemic season. Epidemiologically related isolates (from coinfected twins, from infants infected during a nosocomial outbreak at an extended care facility, and from institutionalized adults infected during an outbreak) yielded identical patterns. In two separate outbreaks, differences in cleavage patterns among certain isolates corresponded to epidemiologically significant differences among the individuals from whom the isolates were recovered. We conclude that substantial genomic heterogeneity exists among circulating isolates of subgroup A RS virus. Ribonuclease protection can be used as a molecular fingerprinting tool for expanded studies of the molecular epidemiology of this virus.
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PMID:RNA fingerprinting of respiratory syncytial virus using ribonuclease protection. Application to molecular epidemiology. 272 64


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